Combined Effects of Different Bracket Bonding Adhesives and Different Resin Removal Methods on Enamel Discoloration: A Preliminary Study

Introduction This study aimed to investigate the effects of 3 orthodontic bracket adhesives and 3 resin removal methods on enamel discoloration. Methods Ninety metal orthodontic brackets were bonded to 90 intact human premolars, using 3 adhesives (total etch composite (Transbond), self-etch composite (OptiBond), and light-curedresin-modified glass ionomer cement (RMGI, Fuji); n = 3 × 30). Each “bracket bonding” group (n = 30) was randomly divided into three subgroups of 10 specimens each, each with a different method of remnant resin removal (using only tungsten carbide burs; using tungsten carbide burs plus Sof-Lex polisher discs; using tungsten carbide burs and Stainbuster burs; n = 3 × 30). After bracket debonding and coffee staining (at 37°C for one week), the color change parameters (Δa, Δb, ΔL, and ΔE) were measured and then analyzed statistically (α = 0.05). Results All 9 mean ΔE values were significantly greater than 3.7 and 1.0 (P values ≤0.002, t-test). The effects of composites and resin removal methods on the ΔE parameter (and their interaction) were significant (P values ≤0.008, two-way ANOVA). There were significant pairwise comparisons between total etch (Transbond) and each of the other composites (P values ≤0.008, Tukey). Nonetheless, the difference between self-etch (OptiBond) and RMGI (Fuji) was not significant (P=0.967). There were significant pairwise comparisons between the ΔE parameter of group “Bur + Stainbuster” and ΔE of each of the other methods (both P values ≤0.017). Conclusions All 9 pairs of adhesives and resin removal techniques will cause quite visible discolorations. Still, self-etch composites or RMGI might be recommended over total etch composites. Moreover, using Stainbuster burs together with tungsten carbide burs is recommended to reduce discoloration. However, the coloration caused by each composite type can change drastically given the following adhesive removal technique used.


Introduction
Orthodontic brackets and their removal may cause damage to the enamel as a direct result of dental enamel manipulation or conditions altered by bonding agents [1]. Bonding and debonding processes may wear of about 10-20% of the enamel surface and cause changes in its appearance [2].
Moreover, the removal of adhesive remnants can change the color of the enamel because the resin irreversibly penetrates the enamel and changes its internal color, not to mention the formation of white spot [11,12]. Te search for an efcient and safe method to remove remnant adhesives has led to the introduction of various techniques for resin removal, which include scraping the surface with a scaler to remove resin, removal with tungsten carbide burs or diamond burs, using polishing discs and paste, air abrasion techniques, and ultrasonic application [13][14][15]. Despite all the studies in this regard, a safe and efective technique for resin removal has not been determined yet. Many dentists try to remove residual resin without sufcient knowledge and based on trial and error, which in some cases can damage the teeth [16]. Even under laboratory conditions, it is virtually impossible to remove the entire adhesive residue from the enamel surface without the aid of high magnifcation [17].
Terefore, the availability of an orthodontic bonding agent with minimal discoloration and the ability to remove its residue by following a simple protocol would be highly desirable. Te literature on the efect of orthodontic bracket bonding/removal on enamel discoloration is scarce and controversial [18,19]. Moreover, no study has simultaneously examined both factors afecting the colorability of teeth after bracket debonding (bonding factors and resin removal factors): all of the few previous studies had either investigated bonding factors or resin removal factors but not both. Besides, no studies have investigated dental discoloration with Bis-GMA composites. Terefore, this study was conducted to simultaneously assess all the factors that could play a role in the colorability of teeth (both in the bonding and debonding stages). Te null hypotheses were the lack of any signifcant diference between the groups of diferent adhesives and diferent resin removal methods or diferent pairs of "adhesives and resin removal methods" in terms of any of the colorimetry parameters.

Materials and Methods
Tis experimental in vitro study was performed on 90 intact human premolars. Te teeth had been extracted solely for orthodontic treatment purposes. Terefore, no harm was done to any individuals. Te ethics of the study were approved by the research committee of the university (ethics code: IR.AJUMS.REC.1399.453). Te inclusion criteria comprised intact buccal enamels free of caries and without any hypocalcifcation or fuorosis on the buccal enamel surface. Te extracted premolars were preserved in a 10% formalin solution until all the teeth were collected.
Similar to all previous studies, there was no mathematical sample size calculation. Instead, we used a rule of thumb and improved it: the sample size for dental colorimetry studies is usually determined as 3 to 6 specimens per group. In this study, this routine number was increased to 10 specimens per group in order to improve the reliability of the fndings. Tere were 9 groups, which accounted for 90 specimens.

Bracket Bonding Groups.
All the experiments were performed according to the manufacturers' instructions. All the brackets used in all 90 specimens were MBT, 0.022" metal brackets (Fairfeld Orthodontics, Fairfeld West Plaza, CA, USA). First, the teeth were randomly divided into 3 "bracket bonding" groups of 30 specimens each. In each group, a diferent adhesive material was used for bonding the metal orthodontic brackets.

Group 1 (Total Etch Composite).
In this group, enamel etching was performed using 37% phosphoric acid for 30 seconds. Ten, the teeth were washed for 10 seconds and sprayed with air for 10 seconds. Bonding of the bracket to the tooth was performed using Transbond MIP Primer adhesive material (3M UNITEK, Monrovia, California, USA) containing primer: TEGDEMA and Bis-GMA and Transbond XT resin composite (3M UNITEK) containing Bis-GMA and TEGDEMA. Tis was performed by applying an adhesive layer on the buccal surface of the tooth and light-curing it for 6 seconds (from a 1 mm distance) using an LED light-curing device (LED.H Ortho, Woodpecker, Beijing, China). Afterward, the composite was placed over the cured adhesive, and the bracket was placed on it. After placing the bracket, the composite resin was light-cured for 20 seconds (10 seconds from the mesial side and 10 seconds from the distal side).

Group 2 (Self-Etch Composite).
In this group, a 7thgeneration bonding agent (self-etching primer, OptiBond All in One, Kerr, CA, USA) was used and no separate etching was done. First, the tooth surface was washed and dried (as mentioned in Group 1). Ten, the self-etch adhesive was scrubbed on the buccal surface of the tooth for 20 seconds. It was then gently air-sprayed for 5 seconds. Ten, it was lightcured for 10 seconds (5 seconds from the mesial side and 5 seconds from the distal side). Next, Transbond XT composite resin (3M Unitek) was used to bond the brackets; after placing the bracket over the smeared composite, it was cured for 20 seconds (as mentioned previously). Ionomer Cement). First, 10% polyacrylic acid was applied for 20 seconds on the tooth surface. Ten, the teeth were washed for 15 seconds, and the tooth surface was kept moist. Ten, light-activated resin-modifed glass ionomer cement (RMGI, Fuji ORTHO LC, GC, Tokyo, Japan) was applied to the enamel as an adhesive material for bonding the brackets. For this purpose, the RMGI adhesive was applied to the buccal surface; then, the bracket was placed on it, and the adhesive was light-cured for 40 seconds (20 seconds from the mesial side and 20 seconds from the distal side).

Bracket Debonding.
After the bonding process, the teeth were kept in artifcial saliva for 48 hours. After that, the brackets were debonded by special orthodontic pliers for bracket removal (straight, Premium-Line, Dentaurum, Ispringen, Germany).

Resin Removal Groups.
In the next step, each of the abovementioned "bracket bonding" groups (n � 30) was randomly divided into three subgroups of 10 specimens each, each with a diferent method of remnant resin removal (or clean-up process). In this way, the whole sample of 90 specimens was divided into 3 "resin removal" groups of 30 specimens each. Te 3 methods of resin removal were 2.3.1. A. Tungsten carbide burs (debonding carbide bur, coarse, Dentaurum) attached to a high-speed handpiece at 40000 rpm, without water irrigation.

B.
Tungsten carbide bur (debonding carbide bur, coarse, Dentaurum) attached to a high-speed handpiece at 40000 rpm, without water spraying, and then polishing the surface with Sof-Lex polisher discs (3M).

Initial Colorimetry.
In order to determine the initial color using the colorimetry parameters (L, a, and b), after resin removal, the teeth were numbered and then photographed with a digital camera (Nikon, Tokyo, Japan), and the images were transferred to the computer. Te images were processed with the help of Photoshop 2018 software (Adobe, San Jose, California, USA), and their primary color was determined. Te "a" parameter is a chromatic coordinate for the green-red spectrum, defned as a spectrum of negative values (green) to positive values (red). Te "b" parameter is a chromatic coordinate for the blue-yellow spectrum, defned as a spectrum of negative values (blue) to positive values (yellow). Te "L" parameter is an indicator of brightness with 0-100 meaning absolutely dark to absolutely bright, respectively.

Cafeine
Staining. Ten, the teeth were stored in a cafeine solution for one week. Te teeth were stored in 100 ml of cafeine solution (Nescafe Classic, Nestle, Switzerland) at 37°C for one week in 9 separate containers (each container for one subgroup of 10). Every day, the cafeine solution was replaced with a new one.

Secondary Colorimetry.
After one week in the cafeine solution, the teeth were cleaned with deionized distilled water. Ten, to evaluate the colorimetry parameters (L, a, and b), a digital photo was taken with a Nikon camera. Te image was evaluated in Photoshop software. To standardize the photography conditions, the teeth were placed in the same position with an indirect light source on a black background and at an angle of 90 degrees to the camera.

Diferential Colorimetry.
Finally, diferential colorimetry was performed by comparing the data obtained from the L, a, and b parameters for the images before and after exposure to cafeine. Te extent of the color diference was calculated using the following formula: ΔE values less than 1 are regarded as color matches; there is a consensus that only ΔE values above 3.7 are considered clinically observable diferences [12,20].

Statistical Analyses.
Descriptive statistics and 95% confdence intervals (CIs) were calculated for various parameters. Data normality was examined and passed using a Kolmogorov-Smirnov test and histogram inspection. A two-way analysis of variance (ANOVA) and a Tukey post hoc test were used to compare the groups with each other in terms of their Δa, Δb, ΔL, and ΔE values. A one-sample t-test was used to compare ΔE values with values 1.0 and 3.7. Since the interactions had become signifcant, subgroup analyses were conducted. As subgroup analyses, a one-way ANOVA followed by a Tukey test was used to compare baseline values as well as Δa, Δb, ΔL, and ΔE values of diferent resin removal methods within each composite. Finally, the one-way ANOVA and Tukey test were used to compare ΔE values within the 9 groups. Te software package in use was SPSS 25 (IBM, Armonk, NY, USA). Te level of signifcance was set at 0.05.

Results
Tere was no missing data. Descriptive statistics and 95% CIs are presented for various subgroups (n � 10), groups (n � 30), and the sample (n � 90) in Tables 1-3 and  Tables 3  and 4. Te pairwise comparisons of 9 pairs of 3 adhesives against 3 resin removal methods are shown in Table 5. Te one-sample ANOVA showed no signifcant comparisons between the baseline values of the subgroups (Table 3).

Delta a (Changes in the Green-Red Spectrum).
Te twoway ANOVA showed a signifcant overall diference among the efects of diferent composites on the Δa parameter (P < 0.00000005, Figures 1 and 2, Tables 1 and 3). Tere was also a signifcant overall diference across the efects of adhesive removal methods on the Δa parameter (P � 0.0000031, Figures 1 and 2, Tables 2 and 3). Te interaction of composites and resin removal methods was as well signifcant, indicating that in diferent composites, the patterns of efects of resin removals may difer (P � 0.0000031, Figure 2, Table 3).

Pairwise Comparisons between Composites.
In terms of composites' efects of the Δa parameter, the Tukey post hoc test showed signifcant pairwise comparisons between RMGI (Fuji) with each of the other composites (both P values ≤0.000003). However, the diference between total etch (Transbond) and self-etch (OptiBond) was insignifcant (P � 0.984, Figures 1 and 2, Table 1).

Pairwise Comparisons between Adhesive Removal
Methods. Te Tukey post hoc test showed signifcant pairwise comparisons between the Δa parameter of group C International Journal of Dentistry    Figures 1 and 2, Table 2).

Subgroup Analyses for Δa (Changes in the Green-Red Spectrum).
According to the one-way ANOVA, the parameter Δa was diferent among the 3 resin removal methods, only within the self-etch composite group (OptiBond, Table 3). Te Tukey test showed that within the self-etch composite (OptiBond), the method of Bur + -Stainbuster had Δa signifcantly diferent from the other resin removal methods (Table 4). However, the other two methods did not difer signifcantly from each other in terms of Δa (Table 4, Figure 2).

Delta b (Changes in the Blue-Yellow Spectrum).
According to the two-way ANOVA, the overall diference across the efects of diferent composites on the Δb parameter was signifcant (P < 0.00000005, Figures 3 and  4, Tables 1 and 3). Similarly, the overall diference among the   efects of resin removal methods on the Δb parameter was signifcant (P � 0.0000007, Figures 3 and 4, Tables 2 and 3). Te interaction of these two variables was signifcant as well (P � 0.0001710, Figure 4, Table 3).

Pairwise Comparisons between Resin Removal
Methods. All the 3 Tukey's pairwise comparisons between the Δb parameters of 3 composites became signifcant (all P values ≤0.014, Figures 3 and 4, Table 2).

Subgroup Analyses for Δb (Changes in the Blue-Yellow
Spectrum). Te parameter Δb was diferent among the 3 resin removal methods, within each of the 3 composites (Table 3). According to the Tukey test, within the total etch     Figure 4).     International Journal of Dentistry parameter was signifcant according to the two-way ANOVA (P � 0.002, Figures 5 and 6, Tables 1 and 3). A signifcant overall diference was as well observed across the infuences of adhesive removal methods on the ΔL parameter (P � 0.011, Figures 5 and 6, Tables 2 and 3). Additionally, the interaction of composites and resin removal methods was signifcant (P � 0.001, Figure 6, Table 3).

Pairwise Comparisons between Resin Removal
Methods. Te pairwise comparisons between the ΔL parameter of group B (Bur + Sof-Lex disc) with the ΔL parameter each of the other methods were signifcant (both P values � 0.025). But the diference between groups A (Bur) and C (Bur + Stainbuster) was insignifcant (P � 0.696, Figures 5 and 6, Table 2).

Subgroup Analyses for ΔL (Brightness Alterations).
Te one-way ANOVA showed that ΔL was diferent among the 3 resin removal methods, only within the self-etch composite group (OptiBond, Table 3). Te Tukey test showed that within the self-etch composite group (Opti-Bond), the only signifcant pairwise comparison was between the ΔL values of the Bur method versus the Bur + Sof-Lex disc (Table 4, Figure 6).

Delta E (the Overall Color Change).
Te efects of different composites on the ΔE parameter were signifcantly diferent (two-way ANOVAs P � 0.002, Figures 7 and 8, Tables 1 and 3). So were the efects of adhesive removal methods on the ΔE parameter (two-way ANOVAs P � 0.001, Figures 7 and 8, Tables 2 and 3) as well as the interaction of composites and adhesive removal methods (P � 0.008, Figure 8, Table 3).

Pairwise Comparisons between Adhesive Removal
Methods. Te Tukey test indicated signifcant pairwise comparisons between the ΔE parameter of group C (Bur + Stainbuster) with ΔE of each of the other methods (both P values ≤0.017). But the diference between ΔE of groups A (Bur) and B (Bur + Sof-Lex disc) was insignifcant (P � 0.500, Figures 7 and 8, Table 2).

Subgroup Analyses for ΔE (Overall Discoloration).
Te parameter ΔE was diferent among the 3 resin removal methods, within 2 of the composites (Table 3). Within the total etch (Transbond) composite group, Bur + Stainbuster had a ΔE signifcantly diferent from the other resin removal methods (Table 4). But the other two methods did not difer signifcantly from each other (Table 4). Within the RMGI (Fuji) composite group, the only signifcant pairwise comparison was between the ΔE values of the Bur method versus the Bur + Sof-Lex disc ( Table 4, Figure 8).

Comparing ΔE Values of 9 Subgroups (Pairs of Adhesives and Resin Removal Methods) with Each Other.
Te one-way ANOVA showed an overall signifcant difference across all the 9 groups (P � 0.00001). Te Tukey test detected 7 pairwise comparisons (Table 5, Figure 8). Te smallest ΔE values were seen when self-etch (OptiBond) or total etch (Transbond) composites were removed using burs together with Stainbuster burs and also when RMGI (Fuji) was removed using Burs + Sof-Lex discs. Te greatest ΔE values were observed when total etch (Transbond) was removed using burs with or without Sof-Lex discs.

Comparing ΔE with the Values 1.0 and 3.7.
Te onesample t-test showed that all the 9 mean ΔE values pertaining to the 9 groups were signifcantly greater than both 3.7 (all the 9 P values ≤0.002) and 1.0 (all the 9 P values <0.0005).

Discussion
Te fndings of the current study showed that all 9 groups caused clinically detectable color changes. Tis was similar to the frst study on the possible changes in enamel color related to orthodontics [12], which showed clinically detectable color alterations (ΔE = 3.7) in all groups [12]. However, their choice of the adhesive system appeared to have insignifcant efects on color changes [12]. Te latter result was in contrast to our fndings, showing that the adhesive in use can afect discoloration extent. Te results of the present study showed that the use of diferent types of adhesives depending on their preparation method can have diferent efects on the colorability of tooth enamel. Te use of RMGI resulted in the lowest amount of colorability, while the use of the 5thgeneration total etch adhesive resulted in the highest amount of colorability. Te observed diference may be attributed to the materials in use and even their brands, the rest of the methodological procedures (e.g., the staining methods, materials, and durations), and even the statistical limitations associated with smaller samples. Another study conducted by Joo et al. [21] on total etch and self-etch adhesives showed that the color change in the conventional acid etching adhesive was greater compared to the SEP adhesive (self-etching primers). A similar result was obtained in the present study. Another study showed no signifcant color change when using self-etch adhesives versus Fuji cement [12]. Tis was in agreement with this research as well. In the present study, the lowest amount of colorability was observed in these two adhesive groups, and no signifcant diference was observed between the two. In the study of Bucar et al. [22], it was shown that color change would be less when using the Fuji cement adhesive compared to the other ones [22], and the same result was obtained in the current study. In a study by Boncuk et al. [23], 2 types of cleaning methods were used to remove adhesive residues in combination with 3 composites. Te greatest color change occurred when only tungsten carbide burs were used. On the other hand, the lowest amount of color alteration was observed when the combination of a tungsten carbide bur and a Stainbuster bur was used [23]. Teir results were consistent with the fndings of this research. Also, their study [23] showed that the use of a Stainbuster bur to remove adhesives had the least efect on enamel discoloration, which resembled the results of the present study.
It was noteworthy that all interactions between all adhesives and all resin removal methods observed in this study were signifcant. Tis means that the results regarding the efects of adhesives on discoloration extents cannot be used without knowing the adhesive removal method, and similarly, the results pertaining to the adhesive removal methods would not be generalizable without specifying the used adhesive. Tis necessitates the assessment of each pair of adhesive plus resin removal methods separately because the overall coloration results may difer for each adhesive depending on the resin removal method to be followed. Hence, adhesive composition along with adhesive removal methods should be investigated in future evaluation for the efect on tooth enamel discoloration. It has been observed earlier that diferent techniques of adhesive removal together with the use of diferent generations and types of adhesive can have diferent efects on enamel discoloration [24]. Te results of the current study showed that the use of the 5th generation adhesive with the total etch preparation method and tungsten carbide bur removal method had the highest amount of color change. Even though the use of 7th generation self-etch adhesive and Fuji cement together with the method of removing resin using tungsten carbide burs and then polishing with Stainbuster burs had the least amount of discoloration on tooth enamel. Ye et al. [24] asserted that the use of the glass-ionomer cement adhesive followed by resin removal using carbide burs and Sof-Lex polishers had the lowest colorability [24]. Very few studies are available in this regard, necessitating more examinations in the future.
On evaluating each colorimetric dimension independently, numerous notable results were obtained. In terms of Δa, it was found that, unlike RMGI which mildly tends to have redder shades, total etch and self-etch composites tend to have greener shades. Using burs with or without Sof-Lex discs would cause greener shades, while adding Stainbuster burs to conventional burs would shift the color to milder redder shades. Regarding Δb, unlike RMGI, which tends to have strong yellow shades, total etch and selfetch composites tend to have bluer shades. All the assessed resin removal methods would cause shades of blue (with Bur + Sof-Lex causing the greatest shades of blue and Bur + Stainbuster causing the mildest shades of blue). Concerning ΔL, RMGI resulted in the brightest ΔL changes, while brightness alterations of total etch and self-etch composites were darker and similar to each other. Moreover, Bur + Sof-Lex resulted in the brightest L changes, while the brightness alterations of Bur and Bur + Stainbuster were darker and similar to each other. Besides, the signifcant interactions would add to the complexity. Te authors could not fnd any other study reporting each dimension of colorimetry independently to compare the results of the present study.
Tis study was limited by some factors. Te sample size was not calculated using mathematical formulas; still, it should be noted that our sample size was much greater than that of many other colorimetry studies that had used a small number of specimens (3 or more) per group. In any study on dental materials, the results of each brand of materials in use might not be generalized to the same types of materials from other brands. Moreover, the in vitro nature of this study necessitates future clinical studies for verifcation of its results in the ever-changing oral environment.
An important point to note is that choosing proper adhesives and resin removal methods depends not only on the discoloration extent caused by these methods but also on other factors including proper bond strengths or enamel damage upon resin removal [4,5,[7][8][9][10]. For example, glass ionomer cements may present signifcantly lower shear bond strengths compared to Transbond XT [10]. Unlike the discoloration caused by orthodontic adhesives and resin removal methods, which is underresearched, factors affecting bracket bond strengths are much more explored and determined [3][4][5][6][7][8][9][10]. Still, future studies are warranted to comparatively investigate the adhesives and resin removal methods presented in this study also in terms of their shear bond strengths and adhesive remnant indexes in order to enable clinicians to decide better.

Conclusions
All 9 pairs of adhesives and resin removal techniques will cause quite visible discolorations. Still, self-etch composites or RMGI might be recommended over total etch composites. Moreover, using Stainbuster burs together with tungsten carbide burs is recommended to reduce discoloration. However, it should be taken into consideration that the coloration caused by each composite type can change drastically given the adhesive removal technique used afterward. Terefore, it is advisable to consider pairs of "composite types together with resin removal methods" instead of relying on the results of composites or resin removal methods alone. In this regard, the composites recommended are self-etch or total etch composites removed by tungsten carbide burs together with Stainbuster burs and also RMGI composites removed using tungsten carbide together with Sof-Lex discs.

Data Availability
Te data are available from the corresponding author upon request.
International Journal of Dentistry